Process for the production of alcohol

ABSTRACT

The invention provides a process for the production of an alcohol which comprises cleaving polysaccharides in a cellulosic material in an aqueous medium at a temperature of at least 45° C. using a thermophilic microorganism to yield fermentable sugars, fermenting an aqueous solution of said sugars at a temperature of at least 45° C. with a thermophilic microorganism to yield an alcohol or an alkanoate, if necessary reducing said alkanoate to yield an alcohol, and removing said alcohol from said aqueous solution.

This invention relates to improvements in and relating to a process forthe production of alcohol from a cellulosic material which may be run atelevated temperature.

Alcohols, or more precisely C₁₋₆ monohydric alkanols, especially ethanoland butanol, are of increasing importance as fuels, either as such or asadditives to conventional hydrocarbon fuels such as gasoline. Alcoholsmay be produced by fermentation of sugars, such as pentoses and hexoses,derived from plant material. While currently much emphasis has been onthe use of plant seeds, e.g. maize, as the raw plant material, this isrelatively undesirable as such seed material may alternatively serve asfood for human or animal consumption. There is thus a desire to useinstead cellulosic material which is unsuitable for human consumption,e.g. wood pulp, forest debris, paper, grass, straw, corn husks, etc. Forsuch raw materials, the cellulose and hemicellulose polysaccharides(hereinafter jointly referred to as cellulose for convenience) mustfirst be broken down to fermentable sugars, generally hexoses andpentoses, whereafter those sugars may be fermented (metabolized) bymicroorganisms to yield alcohols. Yeasts, e.g. brewer's yeast, have longbeen known to be capable of converting the fermentable sugars toalcohols such as methanol and ethanol and bacteria such as Clostridiumhave also long been known to be capable of converting fermentable sugarsto alcohols such as ethanol, propanol and butanol. The alcohols producedmay be separated off from the fermentation mixture (for example bydistillation) and used, e.g. as fuels.

The production of ethanol for use as a fuel in this way has recentlyreceived much attention; however butanol, more specifically n-butanol,is perhaps a more attractive option as a fuel since it is more readilymixed with conventional liquid hydrocarbon fuels and since its calorificyield on combustion is higher than that of ethanol.

The cellulose degradation to fermentable sugars is typically effected byhydrolysis using dilute or concentrated mineral acids, for examplesulphuric or hydrochloric acids. While acid hydrolysis is veryefficient, before the subsequent fermentation step can be carried outthe hydrolysate must be neutralized, e.g. by addition of calciumcarbonate, and the acid and neutralizing base contribute significantlyto the costs of alcohol production from cellulosic starting material.

The fermentable sugars, the product of the polysaccharide (cellulose)degradation, are of course feedstuffs for many if not mostmicroorganisms, including those which do not generate alcohols asmetabolic products. Accordingly it is typically necessary inconventional techniques to sterilize the sugars in order to maximizealcohol production by the alcohol producing microorganism, e.g. thebrewer's yeast.

We have now found that alcohol production from cellulosic raw materialsmay be made more efficient by the use of thermophilic microorganisms ata temperature of at least 45° C., especially at least 50° C.,particularly at least 60° C., e.g. 60 to 80° C., for both cellulose tofermentable sugar degradation and for fermentable sugar to alcoholconversion. In this way the need for sterilization is reduced or avoidedand the need for acid hydrolysis is avoided. Moreover, particularlywhere ethanol or methanol is being produced, the alcohol may bewithdrawn from the fermentation mixture during fermentation so drivingthe fermentation reaction to a higher alcohol yield.

Thus viewed from one aspect the invention provides a process for theproduction of an alcohol which comprises cleaving polysaccharides in acellulosic material in an aqueous medium at a temperature of at least45° C. using a thermophilic microorganism to yield fermentable sugars,fermenting an aqueous solution of said sugars at a temperature of atleast 45° C. with a thermophilic microorganism to yield an alcohol oralkanoate, if necessary reducing said alkanoate to yield an alcohol, andremoving said alcohol from said aqueous solution.

Conversion of fermentable sugars by the microorganism may yield analcohol. Alternatively however, especially where butanol is to beprepared, microorganisms may be used which yield an alkanoate (e.g.butyrate or acetate) instead of or in addition to an alcohol. Thealkanoate can be reduced, e.g. hydrogenated, to form the correspondingalcohol with or without first being separated out of the fermentationmixture. In general however, the processes of the invention willpreferably involve fermentation to yield an alcohol and will not involvealkanoate reduction.

By “thermophilic” is meant herein that the microorganism must be capableof proliferating in aqueous solution at a temperature of at least 45° C.over a prolonged period, e.g. at least 10 hours, preferably atemperature of at least 50° C., more preferably at least 60° C.,especially 60 to 80° C.

The fermentation and cellulose breakdown steps in the process of theinvention are preferably performed at a temperature of least 50° C.,more preferably at least 60° C., especially 60 to 80° C.

In a particularly preferred embodiment of the process of the invention,polysaccharide to fermentable sugar breakdown and fermentable sugar toalcohol (or to alkanoate) conversion are effected in a single stageusing a combination of thermophilic microorganisms. In this embodiment,the different microorganisms may be added to the cellulosic materialsimultaneously or sequentially and in single steps or repeatedly.

Alcohol (or alkanoate) removal from the fermentation medium may beeffected conventionally, e.g. by distillation from the fermentationmedium when fermentation has taken place, or more preferably bywithdrawing the gas from above the fermenting mixture and condensing thealcohol (or alkanoate) from the withdrawn gas. This is especiallypreferred where the alcohol to be produced is methanol or, especially.ethanol. In this embodiment, it is especially preferred to cycle the gasfrom above the fermentation mixture, through a cooled condenser and backinto or to above the fermentation mixture. Apparatus for performing suchan alcohol removal is itself novel and forms a further aspect of theinvention. Thus viewed from this aspect the invention provides apparatusfor alcohol collection comprising: a fermentation vessel having aheater; a condenser; and a gas conduit from said vessel to saidcondenser and back to said vessel. The apparatus is preferably providedwith a pump to facilitate gas flow from the fermentation vessel to thecondenser, with a cooler (e.g. a cooling jacket) for the condenser, andwith an outlet port in the condenser for removal of condensed liquidtherefrom.

Viewed from another aspect the invention provides a process for theproduction of an alcohol (e.g. ethanol, methanol or butanol) whichprocess comprises fermenting an aqueous solution of fermentable sugars(e.g. hexoses and/or pentoses) with a microorganism capable ofconverting said sugars to alcohol in a fermentation vessel at atemperature of 60 to 80° C. under a gaseous atmosphere, and during thefermentation withdrawing gas from said atmosphere into a cooledcondenser to cause entrained alcohol to condense out.

In this process, an inert gas, e.g. nitrogen, hydrogen or carbondioxide, is preferably passed through the fermentation mixture so as toincrease the alcohol content of the gaseous atmosphere removed from thefermentation vessel. This gas may typically be the gas withdrawn fromthe fermentation vessel subsequent to its passage through the condenser.Gas withdrawal moreover moves the alcohol production reactionequilibrium so as to increase alcohol production. Similarly, alcohol maybe removed from the fermentation medium so as to drive the reaction tohigher alcohol production by the use of selective membranes orpervapouration techniques.

The cellulose breakdown to fermentable sugars and the conversion of thefermentable sugars to alcohols according to the invention may be done ina single bioreactor or in a two-step reactor system and themicroorganism or microorganism cocktail used to achieve the conversionsfrom cellulose to sugars and sugars to alcohol may be the same ordifferent.

The microorganisms used for polysaccharide breakdown may be anythermophilic microorganism capable of achieving this. Suitablemicroorganisms may be found in the hot centre of any compost heap.Highly thermophilic microorganisms may be isolated by cultivating asample from such an environment at successively higher temperatures,e.g. raising the temperature in 5 C.° increments from 35° C. to thedesired operating temperatures. Alternatively, and generally morepreferably, such organisms may be isolated from source materials byincubating at the desired, elevated operating temperatures. Examples ofuseful microorganism species include Clostridium thermocellum, C.stercorarum, C. straminisolvens, and C. thermoamylolyticum, especiallyC. thermocellum DSM1237, C. stercorarum DSM8532, C. straminisolvensDSM16021, and C. thermoamylolyticum DSM2335 (see also Ozkan et al., J.Ind. Micribiol. & Biotech. 27:275-280 (2001)).

Suitable cellulose breakdown promoting microorganisms include thoseproducing cellulases, hydrolases, laccases and/or peroxidases.Clostridium strains having cellulose degrading enzymes are known (seefor example Sakka et al, Agricultural and Biological Chemistry53:905-910 (1989), and Kato et al Int. J. Syst. Evol. Microbiol.54:2043-2047 (2004)), and may conveniently be used in the presentinvention.

If desired, the microorganisms used for cellulose breakdown may includeorganisms capable of lignin degradation. Otherwise lignin may be removedfrom the process and used as a fuel to provide part of the energyrequired for the overall process.

The microorganisms used for fermentable sugar to alcohol conversion mayalso be any thermophilic microorganisms capable of achieving this.Thermophilic microorganisms for alcohol production may be identified bycultivating candidates, e.g. yeasts or Clostridium strains at thedesired operating temperatures of the process of the invention, oralternately but less preferably at successively higher temperatures froma lower but elevated temperature up to the desired operatingtemperatures, e.g. raising the temperature in 5 C.° increments from 40°C. to the desired operating temperatures. Thermophilic Clostridiumstrains are already known, e.g. C. thermocellum, C. fervidus, C.thermosulfurogenes, C. thermohydrosulfuricum, C. caminithermale, C.stercorarium, C. thermolacticum, C. thermocopriae, C. straminisolvens,C. thermopapyroliticum, C. thermobutyricum, C. thermopalmarium and C.thermosaccharolyticum (see for example Mendez et al., Int. J. Syst.Bacteriol. 41:281-283 (1991), Jin et al., Int. J. Syst. Bacteriol.38:279-281 (1998), Le Ruyet et al., Syst. Appl. Microbiol. 6:196-202(1985), Madden, Int. J. Syst. Bacteriol. 33:837-840 (1983), Hyun et al.,J. Bacteriol. 156:1332-1337 (1983), Ng et al., Arch. Microbiol. 114:1-7(1977), Wigel et al., J. Ind. Microbiol. and Biotech. 24:7-13 (2000),Lawson et al., Syst. Appl. Microbiol. 14:135-139 (1991), Hollaus et al.,Arch. Micriobiol. 86:129-146 (1972) and McClung, J. Bacteriol.29:189-202 (1935)). One such strain is deposited at the South AmericanBiotechnology and Applied Microbiology Culture Collection as UBA 305.Other microorganism species capable of fermenting at least some of thesugars to form useful alkanols, especially ethanol and butanol, includeThermohydrogenium kirishiense (see Zacharova et al., Arch. Microbiol.160:492-497 (1993)), Thermobacteriodes acetoethylicus (see Ben-Basset etal., Arch. Microbiol. 128:365-370 (1981)), Thermoanaerobiumlactoethylicum (see Kondratieva et al., Arch. Microbiol. 151:117-122(1989)), Butyribacterium methylotrophicum (see Wordet et al., Fuel 70(1990)), and, less preferably, Pyrodictium abyssi (see Pley et al.,Syst. Appl. Microbiol. 14:245-253 (1991)) and Hyperthermus butylicus(see Zillig et al., J. Bacteriol. 172:3959-3965 (1990)).

In a preferred embodiment of the invention, the microorganism used foralcohol production is a genetically modified form of a microorganismcapable of producing an alkanoate from an alkanol bioprecursor, thegenetic modification being to knock out (i.e. disable) or delete a generesponsible for the alkanol bioprecursor to alkanoate conversion. In thecase of Clostridium for example this may involve knocking out ordeleting the gene(s) responsible for converting acetyl-CoA to acetateand/or for converting butyryl-CoA to butyrate or by potentiating orreinforcing the genes responsible for converting acetyl-CoA to ethanolor butyryl-CoA to butanol. This may readily be achieved by conventionaltechniques, such as gene disruption, knock-out mutagenesis or negativeenzyme evolution. Likewise, the microorganism may be transfected with aplasmid capable of generating anti-sense mRNA to block production ofundesired enzymes, e.g. enzymes promoting ethanol production whenbutanol production is desired, and the like. it is also particularlypreferred to utilize a genetically modified form of a microorganismcapable of producing both ethanol and butanol, the genetic modificationbeing to knock out (i.e. disable) or delete a gene responsible for theethanol or the butanol production. In the case of Clostridium forexample this may involve knocking out or deleting the gene(s)responsible for converting acetyl-CoA to ethanol or for convertingbutyryl-CoA to butanol or for converting acetyl-CoA to butyryl-CoA or bypotentiating or reinforcing the genes responsible for convertingacetyl-CoA to ethanol or butyryl-CoA to butanol. This again may easilybe achieved by conventional techniques.

Thus, typically, supplementation of genes for the enzymes acetaldehydedehydrogenase or ethanol dehydrogenase may lead to enhanced ethanolproduction as may deletion, disablement or suppression of the genes forthe enzymes phosphotransacetylase, acetate kinase, thiolase,acetoacetyl-CoA:acetate/butyrate coenzyme-A transferase, acetoacetatedecarboxylase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase,butyryl-CoA dehydrogenase, phosphotransbutyrylase, butyrate kinase,butyraldehyde dehydrogenase, aldehyde/alcohol dehydrogenase E, andbutanol dehydrogenase or disablement of these enzymes or disablement ofRNA coding therefor using antisense RNA. Likewise, supplementation ofgenes for the enzymes butyraldehyde dehydrogenase, aldehyde/alcoholdehydrogenase E, or butanol dehydrogenase, and optionally thiolase,3-hydroxybutyryl-CoA dehydrogenase, crotonase, and butyryl-CoAdehydrogenase may lead to enhanced butanol production as may deletion,disablement or suppression of the genes for the enzymes acetaldehydedehydrogenase, ethanol dehydrogenase, phosphotransacetylase, acetatekinase, acetoacetyl-CoA:acetate/butyrate coenzyme-A transferase,acetoacetate decarboxylase, phosphotransbutyrylase, and butyrate kinaseor disablement of these enzymes or disablement of RNA coding thereforusing antisense RNA.

Suitable starting species for such manipulation to enhance butanolproduction include Clostridium thermobutyricum, C. thermopalmarium, C.thermocopriae, C. thermosaccharolyticum, Eubacterium limosum,Thermohydrogenium kirishiense, Pseudoamibacter alactolyticus,Thermobacteriodes acetoethylicus, Thermoanaerobium lactyloethylicum,Thermoproteus uzoniensis, Pyrodictium abyssi, Hyperthermus butylicus,Thermococcus stetteri and Butyribacterium methylotrophicum.

Thus, for example, butanol production may be enhanced in Clostridium sp.by transformation with plasmids pCAAD or pTHAAD which carry the gene aad(see Nair et al., J. Bacteriol. 176:871-885 (1994) and J. Bacteriol.176:5843-5846 (1994) and Green et al., Biotech. and Bioeng. 58:215-221(1998)). Other plasmids suitable for use in introducing genes into abroad range of Clostridium species are discussed for example by Blascheket al. in FEMS Microbiology Reviews 17:349-356 (1995). Antisense RNA maylikewise be used to suppress the effects of genes which directproduction away from the desired alkanols (see Tummala et al., in J.Bacteriol. 185:1923-1934 (2003)). Classical mutagenesis may of coursealso be used—authors such as Annous et al., in Appl. Env. Microbiol.57:2544-2548 (1991) have reported successful use of classicalmutagenesis in boosting butanol production by Clostridium sp.

Such genetically modified forms of thermophilic alcohol/alkanoateproducing microorganisms are novel and form a further aspect of theinvention. Viewed from this aspect the invention provides a thermophilicmicroorganism, e.g. capable of proliferating at temperatures in excessof 45° C., especially in excess of 50° C., particularly in excess of 60°C., preferably of the species Clostridium, capable of metabolizinghexoses and/or pentoses to produce ethanol and/or butanol wherein a genecoding for an enzyme operative to convert acetyl-CoA to acetate, tobutyryl-CoA or to ethanol or a gene coding for an enzyme operative toconvert butyryl-CoA to butyrate is disabled or deleted. Particularlypreferably, at least two such genes, in particular two or three suchgenes, are disabled or deleted. Disablement or deletion in this contextincludes transformation to generate antisense RNA which reduces orprevents successful gene expression.

Examples of microorganisms useful in the process of the invention forgenerating alcohols or alkanoates, for breaking down biomass to producea substrate for alcohol or alkanoate generation or as starting materialsfor modification as described above include: Clostridium acetobutylicum(grows at 37° C.); C. beijerinckii (grows at 35° C.); C. josui (breaksdown cellubiose, esculin and xylose, grows at 45° C., pH 7.0); C.thermocopriae (breaks down cellulose and a variety of sugars, grows at60° C., pH 6.5-7.3); C. thermosaccharolyticum (breaks down sucrose,dextrin, and pectin, grows at 55-62° C.); C. thermohydrosulfuricum(breaks down starch, cellubiose, glucose, xylose and soluble sugars,grows at 68° C., pH 6.9-7.5); C. thermobutyricum (breaks down solublesugars, grows at 55° C., pH 6.8-7.1); C. thermopalmarium (breaks downsugars, grows at 55° C., pH 6.6); C. carboxidivorans (breaks downglucose, starch, cellulose, cellubiose and pectin, grows at 38° C., pH6.2); Thermobacteroides acetoethylicus (breaks down starch, glucose andother soluble sugars, grows at 65° C., pH 5.5-8.5); Thermoanaerobiumlactoethylicum (breaks down starch, glucose and other sugars, grows at65° C., pH 7.0); Pyrodictium abyssi (breaks down starch and gelatin,grows at 97° C., pH 5.5); Thermococcus stetteri (breaks down peptone,starch and peptin, grows at 73-77° C., pH 6.5); Oxobacter pfennigii(grows at 36-38° C., pH 7.3); Butyribacterium methylotrophicum (grows at37° C., pH 6.0); and Burkholderia xenovorans).

Further examples of microorganisms useful in the process of theinvention for generating butanol or butanoates, for breaking downbiomass to produce a substrate for butanol or butanoate generation or asstarting materials for modification as described above include:Clostridium thermosaccharolyticum ATCC 7956 (butanol-producing, grows at45° C.); C. thermopalmarium DSM 5974 (butyrate-producing, grows at 55°C.); C. carboxidivorans ATCC BAA-624 (butanol-producing, grows at up to40° C.); Thermoanaerobacter acetoethylicus ATCC 33265(butyrate-producing, grows at 60° C.); Thermococcus stetteri DSM 5262(isobutyrate-producing, grows at 75° C.); and Oxobacter pfennigii DSM3222 (butyrate-producing, grows at 37° C.). All of these have an acidgenerating phase followed by a solvent (butanol) generating phase;however the initial butyrate generation is especially effective for C.carboxidivorans, T. acetoethylicus and, especially, O. pfennigii.

In an especially preferred embodiment of the invention, themicroorganisms used for polysaccharide to sugar breakdown and for sugarto alcohol conversion are of the same species, e.g. Clostridium.

Where the microorganisms used are anaerobic, the relevant process stepis preferably performed under an oxygen-free or oxygen-depletedatmosphere (e.g. containing 0 to 10 mole % oxygen, preferably 0 to 5mole %, especially 0 to 2 mole %). In this way competition fornutritional resources by aerobic microorganisms is restricted.Particularly preferably the composition being treated, e.g. an aqueouscellulosic material or an aqueous sugar solution, is treated to reduceoxygen content, for example by exposure to reduced pressure or byflushing with a non-oxygen gas such as nitrogen, carbon dioxide or anoble gas.

As mentioned above, when methanol or ethanol is being produced this maybe removed from the gas above the fermentation mixture duringfermentation. Alternatively, irrespective of the nature of the alcoholbeing produced, the alcohol product may be removed from the fermentedmixture by distillation. In another preferred embodiment, alcohol may beremoved during or after fermentation, preferably during fermentation, bycontacting the aqueous fermentation mixture with a water-immiscibleorganic liquid such as for example a liquid hydrocarbon. The alcohol maybe removed from the organic liquid by distillation or the liquid withentrained alcohol may be used directly as a fuel. Once again, if alcoholis extracted in this way during fermentation, the fermentation reactionmay be driven to increase overall alcohol yield.

The raw starting material for the process of the invention may be anyconvenient cellulosic material. Preferably the material comprises wood(e.g. wood pulp), paper, forest debris, grass, straw, corn husks or thelike. While seeds or nuts as such are not a particularly desirablestarting material, seed or nut waste from pressing for plant oil mayconveniently be used.

Advantageously, the raw material is subjected to chemical and/orphysical pretreatment to accelerate subsequent cellulose breakdown, e.g.maceration or steam treatment.

The invention will now be illustrated further with reference to thefollowing non-limiting Examples and the attached drawing in which:

FIG. 1 is a schematic diagram of apparatus according to the invention.

Referring to FIG. 1, there is shown an apparatus 1 for the production ofan alcohol such as ethanol. Fermentation vessel 2, containingfermentable sugars in aqueous solution 3 produced by cellulosedegradation, is provided with a heating jacket 4 to maintain thesolution temperature at 70±5° C. Conduit 5 leads from vessel 2 tocondenser unit 6 which is provided with a water cooling jacket 7 tomaintain a temperature close to ambient and so cause condensation ofalcohol 8. Return conduit 9 leads back from condenser unit 6 tofermentation vessel 2 via a pump 10. Valve 11 is provided in returnconduit 9 to introduce air or nitrogen or to reduce overpressure in theapparatus as desired or required.

EXAMPLE 1 Polysaccharide Breakdown

Cellulosic material, in this case wood pulp, is preheated by streamexplosion to facilitate subsequent microbial degradation. To thepretreated pulp is added an aqueous inoculate from a compost heap. Themixture is maintained at 60° C. for three days.

EXAMPLE 2 Alcohol Production

The product of Example 1 is inoculated with a butanol producing strainof Clostridium and incubated at 60° C. for two days under a nitrogenatmosphere whereafter the butanol produced is recovered by distillation.

1. A process for the production of an alcohol which comprises cleavingpolysaccharides in a cellulosic material in an aqueous medium at atemperature of at least 45° C. using a thermophilic microorganism toyield fermentable sugars, fermenting an aqueous solution of said sugarsat a temperature of at least 45° C. with a thermophilic microorganism toyield an alcohol, and removing said alcohol from said aqueous solution.2. A process as claimed in claim 1 wherein cleavage and fermentation iseffected at a temperature of at least 60° C.
 3. A process for theproduction of an alcohol which process comprises fermenting an aqueoussolution of fermentable sugars with a microorganism capable ofconverting said sugars to alcohol in a fermentation vessel at atemperature of 60 to 80° C. under a gaseous atmosphere, and during thefermentation withdrawing gas from said atmosphere into a cooledcondenser to cause entrained alcohol to condense out.
 4. A process forthe production of an alcohol which process comprises fermenting anaqueous solution of fermentable sugars with a microorganism capable ofconverting said sugars to alcohol in a fermentation vessel at atemperature of 60 to 80° C. and during the fermentation withdrawingalcohol from said solution.
 5. A process as claimed in claim 1comprising fermenting a said aqueous solution of fermentable sugars witha microorganism capable of converting said sugars to butanol.
 6. Athermophilic microorganism capable of metabolizing hexoses and/orpentoses to produce ethanol and/or butanol wherein a gene coding for anenzyme operative to convert acetyl-CoA to acetate, to butyryl-CoA or toethanol or a gene coding for an enzyme operative to convert butyryl-CoAto butyrate is disabled or deleted.
 7. Apparatus for alcohol collectioncomprising: a fermentation vessel having a heater; a condenser; and agas conduit from said vessel to said condenser and back to said vessel.8. A process as claimed in claim 2 comprising fermenting a said aqueoussolution of fermentable sugars with a microorganism capable ofconverting said sugars to butanol.
 9. A process as claimed in claim 3comprising fermenting a said aqueous solution of fermentable sugars witha microorganism capable of converting said sugars to butanol.
 10. Aprocess as claimed in claim 4 comprising fermenting a said aqueoussolution of fermentable sugars with a microorganism capable ofconverting said sugars to butanol.